PROJECT SUMMARY The lung develops through a series of mesoderm-endoderm interactions that promote proper patterning of the highly complex and arborized structure required for postnatal respiration. We have previously demonstrated the necessity of Wnt signaling in specification of respiratory endoderm through the actions of multiple components of the Wnt pathway including Wnt2/2b and ?-catenin. Later in development, our lab has demonstrated that Wnt signaling is essential for branching point formation in the developing airways, proximal- distal patterning of the developing lung epithelium, and regulating development of smooth muscle lineages within the lung. Thus, Wnt signaling plays multifarious roles in lung development, both in epithelial and mesenchymal lineages. Despite what we have learned about Wnt signaling in lung development, little is known about its role in the adult lung and in particular what role this pathway plays in lung homeostasis or after acute injury. In the mammalian lung, the alveolus is the primary site of gas exchange. Lineage tracing studies have shown that the two primary epithelial lineages of the lung alveolus, alveolar type 1 (AT1) and type 2 (AT2) cells, derive from a common progenitor early in lung endoderm development. Previous studies have also demonstrated that AT2 cells, or a subpopulation within this lineage, can act as a resident progenitor for the adult alveolus through its facultative ability to self-renew and differentiate into AT1 cells after injury. The alveolus also contains a heterogeneous mixture of mesenchymal cells of which little is known. The lack of comparative analysis of the various lung mesenchymal lineages has limited our understanding of the functional cellular heterogeneity of the alveolar niche. Our preliminary studies have defined a progenitor lineage within the overall Sftpc+ cell population, which we have named the alveolar epithelial progenitor (AEP) cell. AEPs express most of the markers of AT2 cells including Sftpc, but unlike the majority of AT2 cells, they also express Axin2, a Wnt signaling target gene and one of the most accurate readouts of Wnt signaling activity. In the adult lung, AEPs comprise approximately 20% of the total AT2 population and using both RNA-seq and ATAC-seq, our unpublished data shows that they have a strikingly distinct transcriptome and epigenome from non-AEP AT2s. In parallel, we have begun to define mesenchymal lineage heterogeneity in the lung using multiple paracrine signaling cell lineage reporters. Using these methods, we have identified a mesenchymal alveolar niche cell (MANC) that are spatially and functionally positioned to promote AT2 self-renewal and differentiation. Together with the characterization of the AEP lineage, we have begun to unravel the cellular and molecular heterogeneity in the lung alveolus. The data from our studies has raised the hypothesis that signaling between distinct mesenchymal (MANCs) and epithelial (AEPs) cells in the alveolus is critical for adult lung homeostasis and regeneration after injury.